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Genetic Diversity of Halophilic organism in Chinese Salt
lakes and application prospect for plant salt resistant
genetic engineering
Fanjing Kong
R & D Center of Salt lake and Epithermal Deposits,CAGS
Main Contents
1. Outline of Chinese salt lakes
2. Genetic diversity of halophilic
organism in Chinese salt lakes
3. Main methods and strategy to
clone genes
Four salt lake regions distinguished:
(I). the Qinghai-Tibet Plateau Region
(II). the Northwestern Region
(III). the North-Central Region
(IV). the East Region
Quaternary saline lake regions in China(After Mianping Zheng,2001)
Characteristics of chemical composition type
1. All types of chemical composition are present in
Chinese salt lakes;
2.From the quaternary arid center of Qaidam BasinTarim Basin outwards the following zones occur: a
chloride-sulphate subtype zone, a chloride-bearing Mgsulphate subtype zone or Na-sulphate zone, and a
carbonate type + Na-sulphate subtype or carbonate type
zone;
3.Some are except which reflect the influence of complex
local geographical and geological conditions
Distribution of salt lakes according chemical types in China
(After Mianping Zheng,1993)
Methods of the genetic diversity study:
Random Amplified Polymorphic DNA
(RAPD)
Restriction Fragmental Length
Polymorphism (RFLP)
DNA Amplified Fingerprinting
(DAF)
Amplified Fragmental Length Polymorphism
(AFLP).
The Genetic diversity of halophilic
bacteria was studied by RFLP
Halophilic bacteria analyzed by16 S r DNA PCR-RFLP
28 moderately halophilic bacteria isolated from salt lakes
in Xinjiang Regions analyzed
The results showed that all tested strains were cluster into
three groups at 74% similarity level.
Group I included two reference strains,
Chromohalobacteria marismortui, Nesternkonia halobia and
new isolate C1.
Group II included seven type strains belonging to
Halomonas and eight new isolates.
Group III included nineteen new isolates
Dendrogram of halophilic bacteria in Xinjiang Region
Table 1 The new halophilic microorganism species from
Chinese Salt lakes
Name
Location
H. dachaidanesis sp. nov.
(F3) and H. Tanggunensis sp.
nov. (F5)
Dachaidam lake in
Qinghai, Tanggu
salt pond in
Tianjing
Dachaidam lake
Halococcus alkalotrophic
dachaidanesis sp. nov
Nb. Chahanesis(C212), Nb.
Chahanesis(X213) and Nb.
Wudunesis(Y212)
H. zangbeiensis sp. nov.
Haloarcula aidinensis sp. nov
New species of Halocuccus
Chahannao lake,
Chagannur,
Wudunao lake in
Inner Mongolia
Zabuye salt lake,
Tibet
Aidin lake in
Xinjiang
Salt lakes in
Xinjiang
Budasun lakes in
qinghai
New alkaliphilic bacterium
species
Chahannao soda
lakein Inner
Mongolia
Natronobacterium
innemongoliae sp. nov
Matai soda lake in
Inner Mongolia
Optimum NaCl
Concentration
18%
Optimu
m pH
Author
Wang D. Z. et al,
1984
18%
8-9
20%
8.7,9.5,8.
7
20%
Wang D. Z. et al,
1986
Wang D. Z. et al,
1989
Zhang J. Z. et al,
1990
Hui S. Y. et al, 1990
25%
Zhou P. J. et al,
1994
Wang D. Z. et al,
1992
3．4M（Na+
2.14M, Mg2+
1.25M）
10.0-11.0%
8.5-12.5
Ma Y. H. et al, 1992
17.5%
9.0-9.5
Tian X. Y. et al,
1997
The genetic diversity of Artemia
was studied by AFLP
Table 2 The Artemia strains for the AFLP analysis
Strains
SX
BD
CG
EJ
TB
AB
BL
GH
DQH
LN
TJ
GL
SF
PS
UM
Species
A．sinica
Location
Yunchen Lake, Shanxi
China
Beidachi, Inner Mongolia,
China
Chagannao, Inner
Mongolia, China
Ejinao Inner Mongolia,
China
Tibet, China
A．tibetiana
A．parthenogenetica Aibi Lake, Xinjiang, China
Balikun, Xingjiang, China
Gahai Lake, Qinghai,
China
Daqinghe, Hebei, China
Yingko, Liaoning, China
Tanggu, Tianjin, China
Great Salt Lake, USA
A．franciscana
San Francisco Bay, USA
Argentina
A．persimilis
Urmia Lake, Iran
A．urmiana
Reproduction model
B
B
B
B
B
P
P
P
P
P
P
B
B
B
B
The result with 12 primer combinations
showed that 594 bands were detected, and
480 bands are polymorphic. The
polymorphic level was 31.819-55.10%.
Average bands for 1 pair are 49.5.
The genetic distance ranged from 0.0690.678, average 0.423
The result demonstrated that the genetic
diversity among Chinese Artemia was very
rich.
Table.3 Genetic distance based on AFLP data of 15
strains of Artemia
A
B
AB
BL
DQ
H
GH
LN
TJ
BD
CG
EJ
GL
PS
SF
SX
UM
TB
BL
0.076
DQH
GH
LN
TJ
BD
CG
EJ
GL
PS
SF
SX
UM
TB
0.540
0.152
0.453
0.56
4
0.31
5
0.40
9
0.37
4
0.56
7
0.52
0
0.46
2
0.43
8
0.37
6
0.39
4
0.525
0.197
0.456
0.628
0.162
0.55
9
0.09
4
0.64
8
0.17
5
0.33
7
0.38
7
0.38
2
0.32
0
0.41
1
0.36
0
0.36
4
0.48
3
0.33
3
0.39
1
0.23
9
0.32
5
0.38
6
0.46
5
0.35
2
0.41
6
0.25
1
0.06
9
0.55
9
0.44
4
0.49
7
0.46
1
0.43
1
0.61
6
0.64
6
0.67
8
0.51
2
0.44
1
0.44
9
0.36
0
0.45
5
0.41
1
0.48
5
0.49
3
0.33
3
0.46
1
0.58
6
0.33
8
0.51
3
0.58
6
0.48
1
0.58
6
0.59
8
0.23
6
0.30
9
0.33
9
0.48
3
0.42
4
0.45
6
0.49
7
0.25
0
0.23
7
0.26
3
0.57
4
0.46
0
0.52
0
0.40
1
0.60
4
0.30
7
0.55
1
0.62
1
0.33
5
0.52
7
0.49
2
0.48
3
0.47
3
0.36
5
0.45
1
0.40
6
0.43
9
0.41
8
0.41
6
0.46
0
0.31
1
0.34
2
0.32
3
0.56
1
0.41
6
0.51
0
0.34
7
0.41
8
0.554
Dendrogram Of Artemia in Chinese salt lakes based
on AFLP data (After Yi Sun, 2000)
The plant genetic engineering for salt resistance
1. Cloning of the salt resistance gene
2. constructs of target gene with vector
3. transformation
4. Identification of transgenetic plants.
The key step is the cloning of salt resistance gene.
The main methods and strategy to
clone gene
Map Based Cloning
Transposon Tagging
Difference Display
Gene Chip
Map Based Cloning
Three main factors should be prepared :
1.high destiny physical maps and
molecular markers linked tightly with
aimed gene
2.clones with large insert DNA fragment
3. efficient transformation system.
The steps of map based cloning
1.identify a molecular marker that lies close
to targeted gene.
2. saturate the region around that original
molecular marker with other markers.
3. screen a large insert genomic library
(BAC or YAC) with your marker to isolate
clones that hybridize to molecular marker.
4.The steps that follow are termed
chromosomal walking.
Differential Display
Differential Display was invented in
1992 by Drs. Arthur Pardee and Peng
Liang to allow rapid, accurate and
sensitive detection of altered gene
expression
Differential Display comparaed with other methods
Gene chip
Traditional methods :a "one gene in one
experiment" basis, throughput is very
limited the, "whole picture" of gene
function is hard to obtain.
Gene chip: promises to monitor the whole
genome on a single chip
a better picture of the interactions among
thousands of genes simultaneously.
Gene chips are fabricated by high-speed
robotics, generally on glass but sometimes
on nylon substrates, for which probes with
known identity are used to determine
complementary binding, thus allowing
massively parallel gene expression and
gene discovery studies.
Summary
1. Most Chinese salt lakes occur in the west
and northeast. They belong to many types
according to their chemical composition.
2. The genetic diversity of halophilic
organisms in salt lake may provide gene
resource for genetic engineering .
3. The strategy and methods for cloning
gene are map based cloning, difference
display, transposon tagging, DNA chip.